It has been shown that eNOS uncoupling occurs in hypertension and atherosclerosis. However its causal role in vascular pathogenesis has not been previously characterized. Here, we challenged eNOS pre-uncoupled hph-1 mice (deficient in eNOS cofactor tetrahydrobiopterin biosynthetic enzyme GTPCHI) with Ang II (0.7 mg/kg/day, 14 days). Both wild-type (WT) and hph-1 groups developed hypertension similarly up to day 6 to 7. Thereafter approximately 14% of Ang II-infused (0.7 mg/kg/day) hph-1 mice (n=72) started to die suddenly of ruptured AAA. Among the survivors, 65% developed AAA, resulting in a total morbidity rate of 79%. In contrast, none of the Ang II-infused WT mice died or developed AAA. Ang II progressively deteriorated eNOS uncoupling in hph-1 mice, while augmenting H4B and nitric oxide (NO•) deficiencies. The abundance of the H4B salvage enzyme dihydrofolate reductase (DHFR) in the endothelium was decreased in hph-1 mice and further diminished by Ang II infusion. Intriguingly, restoration of DHFR expression by oral administration of folic acid (FA), or overexpression of DHFR, completely prevented AAA formation in Ang II-infused hph-1 mice while attenuating progressive uncoupling of eNOS. Folic acid also attenuated vascular remodelling and inflammation characterized by medial elastin break down, augmented MMP2 activity and activation of MMP9, as well as macrophage infiltration. In conclusion, these data innovatively suggest a causal role of eNOS uncoupling/H4B deficiency in AAA formation. Therefore oral FA administration, endothelium targeted DHFR gene therapy, and perhaps other countermeasures directed against eNOS uncoupling, could be used as new therapeutics for AAA.

We have previously shown that eNOS uncoupling mediates abdominal aortic aneurysm (AAA) formation in hph-1 mice. In the present study we examined whether recoupling of eNOS prevents AAA formation in a well-established model of Angiotensin II-infused apolipoprotein E (apoE) null mice by targeting some common pathologies of AAA. Infusion of Ang II resulted in a 92% incidence rate of AAA in the apoE null animals. In a separate group, animals were treated orally with folic acid (FA), which is known to recouple eNOS through augmentation of dihydrofolate reductase (DHFR) function. This resulted in a reduction of AAA rate to 19.5%. Imaging with ultrasound showed that FA markedly inhibited expansion of abdominal aorta. FA also abolished elastin breakdown and macrophage infiltration in the AAA animals. The eNOS uncoupling activity, assessed by L-NAME-sensitive superoxide production, was minimal at baseline but greatly exaggerated with Ang II infusion, which was completely attenuated by FA. This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability. Furthermore, the expression and activity of DHFR was decreased in Ang II-infused apoE null mice specifically in the endothelial cells, while FA administration resulted in its recovery. Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.

Oxidative stress plays an important role in the formation of abdominal aortic aneurysm (AAA), and we have recently established a causal role of uncoupled eNOS in this severe human disease. We have also shown that activation of NADPH oxidase (NOX) lies upstream of uncoupled eNOS. Therefore, identification of the specific NOX isoforms that are required for eNOS uncoupling and AAA formation would ultimately lead to novel therapies for AAA. In the present study, we used the Ang II infused hph-1 mice to examine the roles of NOX isoforms in the development of AAA. We generated double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4. After two weeks of Ang II infusion, the incidence rate of AAA substantially dropped from 76.5% in Ang II infused hph-1 mice (n=34) to 11.1%, 15.0%, 9.5% and 0% in hph-1-NOX1 (n=27), hph-1-NOX2 (n=40), hph-1-p47phox (n=21), and hph-1-NOX4 (n=33) double mutant mice, respectively. The size of abdominal aortas of the four double mutant mice, determined by ultrasound analyses, was significantly smaller than the hph-1 mice. Aortic nitric oxide and H4B bioavailabilities were markedly improved in the double mutants, while superoxide production and eNOS uncoupling activity were substantially diminished. These effects seemed attributed to an endothelial specific restoration of dihydrofolate reductase expression and activity, deficiency of which has been shown to induce eNOS uncoupling and AAA formation in both Ang II-infused hph-1 and apoE null animals. In addition, over-expression of human NOX4 N129S or T555S mutant newly identified in aneurysm patients increased hydrogen peroxide production, further implicating a relationship between NOX and human aneurysm. Taken together, these data indicate that NOX isoforms 1, 2 or 4 lies upstream of dihydrofolate reductase deficiency and eNOS uncoupling to induce AAA formation. These findings may promote development of novel therapeutics for the treatment of the disease by inhibiting NOX signaling.

Obesity is associated with vascular diseases that are often attributed to vascular oxidative stress. We tested the hypothesis that vascular oxidative stress could induce obesity. We previously developed mice that overexpress p22phox in vascular smooth muscle, tgsm/p22phox, which have increased vascular ROS production. At baseline, tgsm/p22phox mice have a modest increase in body weight. With high-fat feeding, tgsm/p22phox mice developed exaggerated obesity and increased fat mass. Body weight increased from 32.16 ± 2.34 g to 43.03 ± 1.44 g in tgsm/p22phox mice (vs. 30.81 ± 0.71 g to 37.89 ± 1.16 g in the WT mice). This was associated with development of glucose intolerance, reduced HDL cholesterol, and increased levels of leptin and MCP-1. Tgsm/p22phox mice displayed impaired spontaneous activity and increased mitochondrial ROS production and mitochondrial dysfunction in skeletal muscle. In mice with vascular smooth muscle–targeted deletion of p22phox (p22phoxloxp/loxp/tgsmmhc/cre mice), high-fat feeding did not induce weight gain or leptin resistance. These mice also had reduced T-cell infiltration of perivascular fat. In conclusion, these data indicate that vascular oxidative stress induces obesity and metabolic syndrome, accompanied by and likely due to exercise intolerance, vascular inflammation, and augmented adipogenesis. These data indicate that vascular ROS may play a causal role in the development of obesity and metabolic syndrome.

Rupture of abdominal aortic aneurysm (AAA) is a lethal event. No oral medicine has been available to prevent or treat AAA. We have recently identified a novel mechanism of eNOS uncoupling by which AAA develops, in Angiotensin II (Ang II) infused hyperphenylalaninemia 1 (hph-1) mice. Using this unique model we investigated effects on AAA formation of the L-type calcium channel blocker nifedipine, in view of the unclear relationship between hypertension and AAA, and unclear mechanisms of aneurysm protective effects of some blood pressure lowering drugs. Six-month old hph-1 mice were infused with Ang II (0.7 mg/kg/day) for 2 weeks, and fed nifedipine chow at two different doses (5 and 20 mg/kg/day). While the high dose of nifedipine reduced blood pressure, the lower dose had no effect. Interestingly, the incidence rate of AAA dropped from 71% to 7 and 12.5% for low and high dose nifedipine, respectively. Expansion of abdominal aorta, determined by ultrasound imaging, was abolished by both doses of nifedipine, which recoupled eNOS completely to improve NO bioavailability. Both also abrogated aortic superoxide production. Of note, Ang II activation of NADPH oxidase in vascular smooth muscle cells and endothelial cells, known to uncouple eNOS, was also attenuated by nifedipine. Although low dose was a sub-pressor while the high dose reduced blood pressure via inhibition of calcium channels, both doses were highly effective in preventing AAA by preserving eNOS coupling activity to eliminate sustained oxidative stress from uncoupled eNOS. These data demonstrate that oral treatment of nifedipine is highly effective in preserving eNOS function to attenuate AAA formation. Nifedipine may be used for AAA prevention either at low dose to AAA risk group, or at high dose to patients with co-existing hypertension.

Hypertension and abdominal aortic aneurysm (AAA) are severe cardiovascular diseases with incompletely defined molecular mechanisms. In the current study we generated dihydrofolate reductase (DHFR) knockout mice for the first time to examine its potential contribution to the development of hypertension and AAA, as well as the underlying molecular mechanisms. Whereas the homozygote knockout mice were embryonically lethal, the heterozygote knockout mice had global reduction in DHFR protein expression and activity. Angiotensin II infusion into these animals resulted in substantially exaggerated elevation in blood pressure and development of AAA, which was accompanied by excessive eNOS uncoupling activity (featured by significantly impaired tetrahydrobiopterin and nitric oxide bioavailability), vascular remodeling (MMP2 activation, medial elastin breakdown and adventitial fibrosis) and inflammation (macrophage infiltration). Importantly, scavenging of mitochondrial reactive oxygen species with Mito-Tempo in vivo completely abrogated development of hypertension and AAA in DHFR knockout mice, indicating a novel role of mitochondria in mediating hypertension and AAA downstream of DHFR deficiency-dependent eNOS uncoupling. These data for the first time demonstrate that targeting DHFR-deficiency driven mitochondrial dysfunction may represent an innovative therapeutic option for the treatment of AAA and hypertension.

Despite an established role of mitochondrial dysfunction in cardiac ischemia reperfusion (I/R) injury, the upstream activators have remained incompletely defined. We have recently identified an innovative role of exogenously applied netrin-1 in cardioprotection, which is mediated by increased nitric oxide (NO) bioavailability. Here, we tested the hypothesis that this “pharmacological” treatment of netrin-1 preserves mitochondrial function via novel mechanisms that are NO dependent. Freshly isolated C57BL6 mouse hearts were perfused using a Langendorff system, and subjected to a 20 min global ischemia/60 min reperfusion, in the presence or absence of netrin-1. I/R induced marked increases in infarct size, total superoxide and hydrogen peroxide production, activity and protein abundance of NADPH oxidase (NOX) isoform 4 (NOX4), as well as impaired mitochondrial integrity and function, all of which were attenuated by netrin-1. This protective effect of netrin-1 is attributed to cGMP, a downstream effector of NO. The protein levels of NOX1 and NOX2 were however unaffected, and infarct size from NOX1 and NOX2 knockouts were not different from wild type animals. Scavenging of NO with PTIO reversed inhibitory effects of netrin-1 on NOX4, while NO donor attenuated NOX4 protein abundance. In vivo NOX4 RNAi, or sepiapterin perfusion, resulted in recoupling of NOS, decreased infarct size, and blockade of dysfunctional mitochondrial swelling and mitochondrial superoxide production. These data demonstrate that netrin-1 induces cardioprotection through inhibition of NOX4 activity, which leads to recoupling of NOS, augmented NO bioavailability, reduction in oxidative stress, and ultimately preservation of mitochondrial function. The NO-dependent NOX4 inhibition connects with our previously established pathway of DCC/ERK1/2/eNOS/NO/DCC feed-forward mechanism, to maintain NOS in the coupling state to attenuate oxidative stress to preserve mitochondrial function. These findings may promote development of novel therapeutics for cardiac I/R injury.

In the present study we aimed to identify novel mechanisms and therapeutics for thoracic aortic aneurysm (TAA) in Fbn1 C1039G/+ Marfan Syndrome (MFS) mice. The expression of mature/active TGFβ and its downstream effector NOX4 were upregulated while tetrahydrobiopterin (H 4 B) salvage enzyme dihydrofolate reductase (DHFR) was downregulated in Fbn1 C1039G/+ mice. In vivo treatment with anti-TGFβ completely attenuated NOX4 expression, restored DHFR protein abundance, reduced ROS production, recoupled eNOS and attenuated aneurysm formation. Intriguingly, oral administration with folic acid (FA) to recouple eNOS markedly alleviated expansion of aortic roots and abdominal aortas in Fbn1 C1039G/+ mice, which was attributed to substantially upregulated DHFR expression and activity in the endothelium to restore tissue and circulating levels of H 4 B. Notably, circulating H 4 B levels were accurately predictive of tissue H 4 B bioavailability, and negatively associated with expansion of aortic roots, indicating a novel biomarker role of circulating H 4 B for TAA. Furthermore, FA diet abrogated TGFβ and NOX4 expression, disrupting the feed-forward loop to inactivate TGFβ/NOX4/DHFR/eNOS uncoupling axis in vivo and in vitro, while PTIO, a NO scavenger, reversed this effect in cultured human aortic endothelial cells (HAECs). Besides, expression of the rate limiting H 4 B synthetic enzyme GTP cyclohydrolase 1 (GTPCHI), was downregulated in Fbn1 C1039G/+ mice at baseline. In cultured HAECs, RNAi inhibition of fibrillin resulted in reduced GTPCHI expression, while this response was abrogated by anti-TGFβ, indicating TGFβ-dependent downregulation of GTPCHI in response to fibrillin deficiency. Taken together, our data for the first time reveal that uncoupled eNOS plays a central role in TAA formation, while anti-TGFβ and FA diet robustly abolish aneurysm formation via inactivation of a novel TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ feed-forward pathway. Correction of fibrillin deficiency is additionally beneficial via preservation of GTPCHI function.